1. Field of the Invention:
This invention relates to the freezing, preservation, storage and transport of samples such as biological samples. In one of its more particular aspects, this invention relates to the rapid freezing of tissue samples for cryostat microtoming and subsequent histopathologic examination. In another aspect, this invention relates to the transport of biological samples under controlled temperature conditions.
In order to examine surgically removed tissues microscopically for the diagnosis of various pathological conditions, it is often necessary to use thin frozen tissue sections. Such thin tissue sections are usually prepared by freezing a tissue sample and then slicing sections from the frozen tissue, for example, by use of a cryostat microtome. Other biological samples frequently must be frozen for purposes of preservation or transport thereof. In addition, low temperature freezing techniques are applicable to nonbiological systems for testing the physical properties of a wide variety of materials.
Advances in frozen section techniques have not paralleled technical advances in every other field of medicine. With each surgical case, pathologist and surgeon will commonly waste critical surgical time waiting for a microscopic diagnosis.
One of the rate limiting factors in microscopic diagnosis is the freezing process. In an attempt to improve upon this frustrating situation, pathologists have tried a wide variety of microtome adaptions and accessories, such as microtome "freezing bars", with limited success.
Low temperature freezing can be accomplished in a number of ways, such as by use of a refrigerator or freezer capable of reducing the temperature of the tissue sample below its freezing point, by spraying the tissue sample with gaseous carbon dioxide or other gas capable of freezing the sample, or by immersing the tissue sample in a suitable mixture of nonaqueous liquid and solid carbon dioxide (dry ice). Each of these methods can be used more or less successfully, but each has its disadvantages.
Use of a refrigerator or freezer is relatively slow. Spraying with a gas is disruptive to the carrying media as well as the tissue and causes irregular freezing of the sample. Additionally, spraying with a gas produces an aerosol effect which is undesirable when working with contaminated or infected tissue specimens. Gaseous CCl.sub.2 F.sub.2 is potentially toxic and has been implicated as a health hazard and as an environmental pollutant.
Immersion of the sample in a solid CO.sub.2 -liquid mixture requires the handling of dry ice. Dry ice is depletable, and a continuous source of dry ice is required for subsequent preparations. Probably the most undesirable feature of the dry ice-liquid mixture technique is the violent effervescence which occurs when the dry ice is introduced into the liquid, producing subsequent artifacts of irregular freezing in the carrying media, as well as in the tissue.
In summary, current methodologies are lengthy, time consuming and labor intensive. Additionally, these methods induce various types of mechanical distortion as well as other types of tissue artifact.
2. Prior Art:
Various frozen tissue techniques are described in Waldemar A. Schmidt, Principles and Techniques of Surgical Pathology, pp. 117-138 and 145-148, Addison Wesley Publishing Co., Inc. (1983). In particular, this reference describes (at p. 123) the cryostat technique which utilizes a rotary microtome within a cabinet refrigerated to about -20 degrees Celsius. This reference also mentions certain rapid freezing techniques such as the use of liquid nitrogen or isopentane cooled in liquid nitrogen, a slush of dry ice and acetone, the Cryokwik Spray (CCl.sub.2 F.sub.2) or a freezing attachment using CO.sub.2 (p. 128).
U.S. Pat. No. 2,067,676 describes a cooling system which utilizes solid carbon dioxide and a liquid for cooling a body immersed in the liquid.
U.S. Pat. No. 3,176,472 describes a microtome freezing system utilizing thermoelectric freezing by means of a salt brine bath.
U.S. Pat. No. 3,360,943 describes the use of a mixture of dry ice with a liquid such as acetone for the purpose of mechanical testing of metal specimens.
U.S. Pat. No. 3,828,571 describes a particular construction of microtome.
U.S. Pat. No. 3,871,107 describes a freeze dryer which utilizes an aluminum container cooled by snow formed upon the expansion of carbon dioxide gas into the container.
U.S. Pat. No. 3,975,977 describes a microtome with an automatic knife-lowering mechanism.
U.S. Pat. No. 4,004,975 describes a method for cryopreserving human white cells by using a combination of hydroxeythyl starch and dimethyl sulfoxide.
U.S. Pat. No. 4,008,754 describes the use of an inert gas to freeze organs for conservation.
U.S. Pat. No. 4,199,954 describes a method of freezing cells by using a mixture of uranyl acetate and ethyl alcohol.
U.S. Pat. No. 4,224,801 describes a refrigeration unit which is cooled by dry ice mixed with liquid carbon dioxide in a slush tank.
U.S. Pat. No. 4,374,658 describes a machine for making block dry ice.